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R W Davis

Publications and source records attributed to R W Davis.

At least 55 records · Page 3Linked to original sources
PubMed1999

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation.

The recA gene of Escherichia coli is the prototype of the recA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination. In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants. Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system. All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage. The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover. Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA-independent, lethal filamentation of cells.

Adenosine Triphosphate↗
PubMed1999

On the in vivo function of the RecA ATPase.

The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.

Adenosine Triphosphatases↗
PubMed1999

An arrayable flow-through microcentrifuge for high-throughput instrumentation.

A compact, flow-through centrifugation system has been developed specifically for high-throughput centrifugation of large numbers of samples. The instrument is based on multiple high-speed rotors that also serve as sample holders. The small size of the rotors allows them to be arrayed in a standard 96-well microtiter plate spacing, making this device ideal for highly parallel automated instrumentation. Though initially designed for cell separation in DNA sequencing protocols, the flow-through centrifuge can be used to replace conventional centrifugation in most processes involving small samples. Techniques for recovery of both the supernatant and the pellet have been developed, as well as techniques for sample mixing, and cleaning of the reusable rotors. This paper discusses the design and performance of the flow-through centrifuge applied to cell separation and resuspension and to DNA purification and concentration.

Centrifugation↗
PubMed1999

Whole genome genetic-typing in yeast using high-density oligonucleotide arrays.

Genome sequence information in combination with new technologies has allowed researchers to approach genetic problems in new ways. High-density oligonucleotide arrays were used to probe the genome content of the yeast Saccharomyces cerevisiae. We show that these arrays, containing oligonucleotides complementary to the sequenced strain of S. cerevisiae, can be used to identify open reading frames that are missing or present in higher or lower copy number in related isolates of S. cerevisiae. We apply this method to the characterization of the genome of a strain derived from a clinical isolate of S. cerevisiae. Our results show that the telomeres are the regions with the most variability between the two strains.

Gene Deletion↗
PubMed1999

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Arabidopsis↗
PubMed1999

Genomic profiling of drug sensitivities via induced haploinsufficiency.

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

Anti-Bacterial Agents↗
PubMed1999

Comparative genomes of Chlamydia pneumoniae and C. trachomatis.

Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

Amino Acid Sequence↗
PubMed1999

Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen. Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays. This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate. Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions. Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions.

Aerobiosis↗
PubMed1999

High aerobic capacities in the skeletal muscles of pinnipeds: adaptations to diving hypoxia.

The objective was to assess the aerobic capacity of skeletal muscles in pinnipeds. Samples of swimming and nonswimming muscles were collected from Steller sea lions (Eumetopias jubatus, n = 27), Northern fur seals (Callorhinus ursinus, n = 5), and harbor seals (Phoca vitulina, n = 37) by using a needle biopsy technique. Samples were either immediately fixed in 2% glutaraldehyde or frozen in liquid nitrogen. The volume density of mitochondria, myoglobin concentration, citrate synthase activity, and beta-hydroxyacyl-CoA dehydrogenase was determined for all samples. The swimming muscles of seals had an average total mitochondrial volume density per volume of fiber of 9.7%. The swimming muscles of sea lions and fur seals had average mitochondrial volume densities of 6.2 and 8.8%, respectively. These values were 1.7- to 2.0-fold greater than in the nonswimming muscles. Myoglobin concentration, citrate synthase activity, and beta-hydroxyacyl-CoA dehydrogenase were 1.1- to 2. 3-fold greater in the swimming vs. nonswimming muscles. The swimming muscles of pinnipeds appear to be adapted for aerobic lipid metabolism under the hypoxic conditions that occur during diving.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

The diving physiology of bottlenose dolphins (Tursiops truncatus). II. Biomechanics and changes in buoyancy at depth.

During diving, marine mammals must balance the conservation of limited oxygen reserves with the metabolic costs of swimming exercise. As a result, energetically efficient modes of locomotion provide an advantage during periods of submergence and will presumably increase in importance as the animals perform progressively longer dives. To determine the effect of a limited oxygen supply on locomotor performance, we compared the kinematics and behavior of swimming and diving bottlenose dolphins. Adult bottlenose dolphins (Tursiops truncatus) were trained to swim horizontally near the water surface or submerged at 5 m and to dive to depths ranging from 12 to 112 m. Swimming kinematics (preferred swimming mode, stroke frequency and duration of glides) were monitored using submersible video cameras (Sony Hi-8) held by SCUBA divers or attached to a pack on the dorsal fin of the animal. Drag and buoyant forces were calculated from patterns of deceleration for horizontally swimming and vertically diving animals. The results showed that dolphins used a variety of swimming gaits that correlated with acceleration. The percentage of time spent gliding during the descent phase of dives increased with depth. Glide distances ranged from 7.1+/-1.9 m for 16 m dives to 43.6+/-7.0 m (means +/- s.e.m.) for 100 m dives. These gliding patterns were attributed to changes in buoyancy associated with lung compression at depth. By incorporating prolonged glide periods, the bottlenose dolphin realized a theoretical 10-21 % energetic savings in the cost of a 100 m dive in comparison with dives based on neutral buoyancy models. Thus, modifying locomotor patterns to account for physical changes with depth appears to be one mechanism that enables diving mammals with limited oxygen stores to extend the duration of a dive.

Animals↗
PubMed1999

Convective oxygen transport and tissue oxygen consumption in Weddell seals during aerobic dives.

Unlike their terrestrial counterparts, marine mammals stop breathing and reduce their convective oxygen transport while performing activities (e.g. foraging, courtship, aggressive interactions, predator avoidance and migration) that require sustained power output during submergence. Since most voluntary dives are believed to remain aerobic, the goal of this study was to examine the potential importance of the dive response in optimizing the use of blood and muscle oxygen stores during dives involving different levels of muscular exertion. To accomplish this, we designed a numerical model based on Fick's principle that integrated cardiac output (Vb), regional blood flow, convective oxygen transport (Q(O2)), muscle oxymyoglobin desaturation and regional rates of oxygen consumption (VO2). The model quantified how the optimal matching or mismatching of QO2 to VO2 affected the aerobic dive limit (ADL). We chose an adult Weddell seal Leptonycotes weddellii on which to base our model because of available data on the diving physiology and metabolism of this species. The results show that the use of blood and muscle oxygen stores must be completed at the same time to maximize the ADL for each level of VO2. This is achieved by adjusting Vb (range 19-94 % of resting levels) and muscle QO2 according to the rate of muscle oxygen consumption (VMO2). At higher values of VMO2, Vb and muscle perfusion must increase to maintain an appropriate QO2/VO2 ratio so that available blood and muscle oxygen stores are depleted at the same time. Although the dive response does not sequester blood oxygen exclusively for brain and heart metabolism during aerobic dives, as it does during forced submersion, a reduction in Vb and muscle perfusion below resting levels is necessary to maximize the ADL over the range of diving VO2 (approximately 2-9 ml O2 min-1 kg-1). Despite the reduction in Vb, convective oxygen transport is adequate to maintain aerobic metabolism and normal function in the splanchnic organs, kidneys and other peripheral tissues. As a result, physiological homeostasis is maintained throughout the dive. The model shows that the cardiovascular adjustments known as the dive response enable the diving seal to balance the conflicting metabolic demands of (1) optimizing the distribution and use of blood and muscle oxygen stores to maximize the ADL over the normal range of diving VO2 and (2) ensuring that active muscle receives adequate oxygen as VMO2 increases.

Aerobiosis↗
PubMed1999

An automated sample preparation system for large-scale DNA sequencing.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

DNA↗
PubMed1998

Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.

Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.

Aerobiosis↗
PubMed1998

Direct allelic variation scanning of the yeast genome.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

Alleles↗
PubMed1998

Parallel analysis of genetic selections using whole genome oligonucleotide arrays.

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

Animals↗
PubMed1998

Microarrays: biotechnology's discovery platform for functional genomics.

Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale. Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction. These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness. Microarray technology is rapidly becoming a central platform for functional genomics.

Biotechnology↗
PubMed1998

A genome-wide transcriptional analysis of the mitotic cell cycle.

Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

Cell Cycle↗